Methods of Generating Mature Human Muscle Fibers

ABSTRACT

The invention relates to methods of treating a subject having a muscle disorder by identifying a subject having a muscle disorder in need of treatment; injecting human myogenic precursor cells in an amount capable of forming mature muscle tissue into a portion of a limb of the subject; subjecting a nerve of the limb to therapeutic stimulation configured to enhance engraftment of the human myogenic precursor cells; and creating a graft of the human myogenic precursor cells to promote generation of mature muscle tissue, wherein the generation of mature muscle tissue improves muscle function. Preferably, the subject is a mammal, such as a human or a non-human mammal. In some embodiments, the non-human mammal is immunocompromised and the limb is irradiated. The engraftment can be promoted by a means other than therapeutic electrical stimulation (preferably intermittent neuromuscular electrical stimulation), for example by exercise.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/029,191, which is a 371 national stage application of PCTApplication No. PCT/US2014/061262, filed Oct. 17, 2014, and claims thebenefit of Provisional application 61/892,104, filed Oct. 17, 2013,under 35 U.S.C. § 119(e), the entire contents of each application arehereby incorporated by reference as if fully set forth herein.

GOVERNMENT FUNDING SUPPORT

This invention was made with government support under Grant Nos.HD060848 and NS086902 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Muscular dystrophies afflict approximately 1 in 5000 individualsworldwide. The development of animal models to study these diseases isessential in understanding the mechanisms of pathogenesis and to testpotential therapies for people afflicted with these diseases. Animalmodels for some muscular dystrophies, such as facioscapulohumeralmuscular dystrophy (“FSHD”) and oculopharyngeal muscular dystrophy(“OPMD”), are still unavailable. Furthermore, some murine models of somemuscular dystrophies, whether naturally occurring or geneticallyengineered, are limited because they do not replicate all the featuresof the human disease.

An animal model that carries human muscles from individuals with thesediseases would serve as a more accurate model, reproducing most, if notall, of the morphological, physiological and genomic features of themuscular dystrophies in man. The present invention describes methods todevelop such a model, while solving the problem of the presence ofmurine myonuclei in the graft. Here, intermittent neuromuscularelectrical stimulation (iNMES) is applied to immunodeficient miceengrafted with an immortalized clonal cell line of human myogenicprecursor cells (“hMPCs”) that express luciferase (“LHCN” cells).

Previous studies of grafts of hMPCs create hybrid fibers, containingmyonuclei of both human and murine origin form, and the fibers expressproteins found both in man and in mice. In the present invention, muscleof the host mouse was eliminated by injection of a myotoxin and themyogenic potential of any remaining tissue was suppressed byX-irradiation. Human myogenic precursor cells were then injected. Thesurvival and differentiation of the human muscle tissue were promotedwith electrical stimulation. Many of the myofibers in that graft weresimilar to nearby murine myofibers in size, and they were bothinnervated by motor neurons and fully differentiated. More importantly,they were comprised almost exclusively of human myonuclei, with minimalcontamination by murine myonuclei.

SUMMARY OF THE INVENTION

It has been discovered that muscle formed from transplanted LHCN cellsis highly differentiated and innervated, and is composed of myofibers ofhuman origin, with minimal contamination by murine myonuclei. It hasalso been discovered that neuromuscular electrical stimulation (“NMES”)enhances engraftment of these immortalized human myoblasts in animals.

In certain embodiments, methods are provided for treating a subjecthaving a muscle disorder. First, a subject having the muscle disorder isidentified. Human myogenic precursor cells in an amount capable offorming mature muscle tissue are injected into a portion of a limb ofthe subject. A nerve of the limb is then subjected to a regime oftherapeutic stimulation (e.g., NMES) configured to promote and enhanceengraftment of the human myogenic precursor cells. A graft is createdand promotes formation of mature muscle tissue that improves thefunction of muscle. In some embodiments, the subject is a non-humanmammal, e.g., a mouse. If the subject is non-human, it must beimmunodeficient, and thus incapable of mounting an immune response toinjected human cells and the subsequent graft.

In some aspects, the invention further provides a method for producing anon-human animal that models a human muscular disease. A non-humananimal such as an immunocompromised mouse suitable for xenografting isobtained. The limb (e.g., a hindlimb) of the non-human animal isirradiated by exposure to with X-rays or other form of irradiation knownin the art. Muscle (i.e., the Tibialis anterior muscle “TA”) of thatlimb was then injected along its length with a toxin such ascardiotoxin, a phospholipase that causes the muscle to degenerate. hMPCscapable of forming muscle tissue were injected into the compartmentformerly occupied by the degenerated muscle tissue and then the peronealnerve serving that compartment was subjected to electrical stimulationto enhance engraftment of the myoblast cells so that they develop intomature muscle tissue. The engraftment of the myogenic precursor cells inthe limb treated with a regime of electrical stimulation, such as NMES,was improved compared to engraftment of a population of untreatedmyoblast cells. The myogenic precursor cells can be derived from healthyhumans or from individuals with certain muscle diseases, notablyFacioscapulohumeral Muscular Dystrophy (FSH or FSHD). Non-human animalmodels generated by this method in animals other than mice are alsocontemplated.

Still other aspects, features, and advantages of the invention arereadily apparent from the following detailed description, simply byillustrating a number of particular embodiments and implementations,including the best mode contemplated for carrying out the invention. Theinvention is also capable of other and different embodiments, and itsseveral details can be modified in various obvious respects, all withoutdeparting from the spirit and scope of the invention. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

The present invention relates to a method of treating a subject having amuscle disorder comprising the steps of (a) identifying a subject havinga muscle disorder in need of treatment; (b) injecting human myogenicprecursor cells in an amount capable of forming mature muscle tissueinto a portion of a limb of the subject; (c) subjecting a nerve of thelimb to therapeutic stimulation configured to enhance engraftment of thehuman myogenic precursor cells; and (d) creating a graft of the humanmyogenic precursor cells to promote generation of mature muscle tissue,wherein the generation of mature muscle tissue improves muscle function.Preferably, the subject is a mammal, such as a human or a non-humanmammal. In some embodiments, the non-human mammal is immunocompromisedand the limb is irradiated. The therapeutic stimulation can betherapeutic electrical stimulation. The engraftment can be promoted by ameans other than therapeutic electrical stimulation (preferablyintermittent neuromuscular electrical stimulation), for example byexercise. The muscle disease can be selected from the group consistingof: acid maltase deficiency (AMD), Andersen-Tawil Syndrome, BeckerMuscular Dystrophy (BMD), Becker Myotonia Congenita, Bethlem Myopathy,Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy), CarnitineDeficiency, Central Core Disease (CCD), Centronuclear Myopathy,Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD),Congenital Myotonic Dystrophy, Dejerine-Sottas Disease (DSD),Dermatomyositis (DM), Distal Muscular Dystrophy (DD), Duchenne MuscularDystrophy (DMD), Dystrophia Myotonica (Myotonic Muscular Dystrophy),Emery-Dreifuss Muscular Dystrophy (EDMD), Eulenberg Disease(Paramyotonia Congenita), Facioscapulohumeral Muscular Dystrophy (FSH orFSHD), Finnish (Tibial) Distal Myopathy, Friedreich's Ataxia (FA),Fukuyama Congenital Muscular Dystrophy, Glycogenosis Type 2,Glycogenosis Type 5, Glycogenosis Type 7, Glycogenosis Type 9,Gowers-Laing Distal Myopathy, Hauptmann-Thanheuser MD (Emery-DreifussMuscular Dystrophy), Hereditary Inclusion-Body Myositis, HereditaryMotor and Sensory Neuropathy (Charcot-Marie-Tooth Disease), HyperthyroidMyopathy, Hypothyroid Myopathy, Inclusion-Body Myositis (IBM), InheritedMyopathies, Integrin-Deficient Congenital Muscular Dystrophy, KennedyDisease (Spinal-Bulbar Muscular Atrophy), Kugelberg-Welander Disease(Spinal Muscular Atrophy), Lactate Dehydrogenase Deficiency,Lambert-Eaton Myasthenic Syndrome (LEMS), Limb-Girdle MuscularDystrophies (LGMDs), Lou Gehrig's Disease (Amyotrophic LateralSclerosis), Merosin-Deficient Congenital Muscular Dystrophy, MetabolicDiseases of Muscle, Mitochondrial Myopathy, Miyoshi Distal Myopathy,Motor Neurone Disease, Muscle-Eye-Brain Disease, Myasthenia Gravis (MG),Myofibrillar Myopathy, Myotonia Congenita (MC), Myotonic MuscularDystrophy (MMD), Myotubular Myopathy (MTM or MM), Nemaline Myopathy,Nonaka Distal Myopathy, Oculopharyngeal Muscular Dystrophy (OPMD),Paramyotonia Congenita, Periodic Paralysis, Peroneal Muscular Atrophy(Charcot-Marie-Tooth Disease), Pompe Disease (Acid Maltase Deficiency),Progressive External Ophthalmoplegia (PEO), Rod Body Disease (NemalineMyopathy), Spinal Muscular Atrophy (SMA), Spinal-Bulbar Muscular Atrophy(SBMA), Steinert Disease (Myotonic Muscular Dystrophy), Thomsen Disease(Myotonia Congenita), Ullrich Congenital Muscular Dystrophy,Walker-Warburg Syndrome (Congenital Muscular Dystrophy), Welander DistalMyopathy, and Werdnig-Hoffmann Disease (Spinal Muscular Atrophy).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1A-1D are representative BLI images of NOD-RAG mice on days 0, 7,28, and 49 after injection with 5×10⁶ or with 2×10⁶ LHCN cells andsubjected to 4 weeks of NMES, according to an embodiment.

FIG. 2 is a graph illustrating quantitative analysis of bioluminescenceas a function of time following transplantation, according to anembodiment.

FIG. 3A-3B are fluorescent images illustrating human muscle fibers inengrafted limbs collected at 4 weeks after injection of 2.5×10⁵ LHCNcells, according to an embodiment.

FIG. 4 is a graph illustrating the number of muscle fibers of humanorigin per graft after injection of 5×10⁵ and 2.5×10⁶ LHCN cells,according to an embodiment.

FIG. 5 is a graph illustrating the sizes of human myofibers in graftsafter injection of 5×10⁵ LHCN cells, 2.5×10⁶ LHCN cells, and 2.5×10⁶LHCNcells plus NEMS, according to an embodiment.

FIG. 6 is a graph illustrating the distances between the largest fibersof human origin in each graft and each of their nearest neighbors ofhuman origin after injection of 2.5×10⁶ cells and 2.5×10⁶ cells plusNMES, according to an embodiment.

FIG. 7A-7B are fluorescent images illustrating the presence of collagenand fibrosis in human muscle grafts after injection of 2×10⁶ LHCN cellsand 2×10⁶ LHCN cells plus NMES, according to an embodiment.

FIG. 8 is a fluorescent image illustrating organization of desmin infibers of human origin after injection of 2×10⁶ LHCN cells and 2×10⁶LHCN cells plus NMES, according to an embodiment.

FIG. 9A-9B are fluorescent images illustrating quantitation of centralnuclei of human and mouse origin in fibers of human origin, according toan embodiment.

FIG. 10A-10B are fluorescent images illustrating neuromuscular junctionsin grafts treated with iNMES, according to an embodiment.

FIG. 11 is a flow chart that illustrates an example method forgenerating human muscle fibers, according to an embodiment.

DETAILED DESCRIPTION

One or more methods are described for (i) enhancing engraftment ofmyogenic precursor cells; (ii) generating mature muscle fibers; (iii)treating muscle disorders and (iv) producing non-human animals thatmodel muscle disease e.g., FSHD. In the following description, for thepurposes of explanation, numerous specific details are set forth inorder to provide a thorough understanding of the present invention. Itwill be apparent, however, to one skilled in the art that the presentinvention may be practiced without these specific details. In otherinstances, well-known structures and devices are shown in block diagramform in order to avoid unnecessarily obscuring the present invention.Various references are cited in the following. The entire contents ofthe following references are hereby incorporated by reference as iffully set forth herein, except for terminology that is inconsistent withthe terminology used herein.

Some embodiments of the invention are described below in the context ofa mouse model that has a muscle disease. However, the invention is notlimited to this context. In other embodiments the subject is a humanbeing or other animal.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The following terms as used herein have the corresponding meanings givenhere.

1. DEFINITIONS

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise. As used inthis specification and the appended claims, the terms “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

As used herein, the terms “animal,” “patient,” or “subject,” mean anyanimal (e.g., mammals, including but not limited to humans, primates,dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats,rabbits, rodents, and transgenic non-human animals) that is to be therecipient of a particular treatment. Typically, the terms “animal,”“subject,” and “patient” are used interchangeably herein in reference toa human subject or a rodent. The preferred animal, patient, or subjectis a human or a non-human mammal such as a mouse.

As used herein, the term “muscle disorder” is intended to broadlyencompass muscle disease, muscle injuries, and muscle disorders, anddefects that can impair and reduce muscle function including but notlimited to physical injuries, burns, surgical tissue excisions, musclewasting, muscular dystrophy, infarcts, ischemic events, neuromusculardisorders and muscle diseases including, but not limited to those, setforth in Table 1.

As used herein, “muscle cells” include, but are not limited to, skeletalmuscle fibers, myofibers or myocytes, and may be of any suitablespecies, and in some embodiments are of the same species as the subjectinto which tissues are implanted. Mammalian cells (including mouse, rat,dog, cat, monkey and human cells) are in some embodiments particularlypreferred.

As used herein, the term “muscle fiber” or “myofiber” refers to amultinucleated single muscle cell. Physically, e.g., in humans, they arehighly elongated and are typically 50-100 microns in diameter, but rangein length from a few millimeters many centimeters. Muscle fiber cellsare formed from the fusion of myoblasts (a type of progenitor cell thatgives rise to a muscle cell during development or, in adults, duringregeneration following injury). The myofibers are long, cylindrical,multinucleated cells composed of actin and myosin myofibrils repeated asa sarcomere, the basic functional unit of the muscle fiber andresponsible for skeletal muscle's striated appearance and forming thebasic machinery necessary for muscle contraction.

As used herein, the term “myoblasts” are a type of muscle precursorcell. They are present in developing muscle and appear in adult musclewhen satellite cells, muscle stem cells that are closely associated withmyofibers in vertebrates, become activated. If the myofiber is injured,the myoblasts are capable of dividing and fusing to form a new myofiber.Typically, after muscle injuries, myofibers become necrotic and areremoved by macrophages. This induces activation of stem cells and theproliferation and fusion of myoblasts to form multinucleated andelongated myotubes, which develop further into myofibers. The myofibersso generated form a more organized structure, namely muscle.

As used herein, the term “myocytes” are muscle cells, muscle fibers, orskeletal muscle cells, in either the mature or immature state.

As used herein, the term, “myofibrils” are the slender threads of amuscle fiber composed of numerous myofilaments. Myofibrils run from oneend of the cell to the other and attach to the cell surface membrane ateach end.

As used herein, the term “myotubes” are elongated, multinucleated cells,normally formed by the fusion of myoblasts. Myotubes have centrallylocated nuclei and myofibrils that tend to be poorly organized. Invertebrates, they develop into mature muscle fibers, which haveperipherally-located nuclei and myofibrils that are well organized(e.g., in mammals). Under low serum conditions, myoblasts exit the cellscycle and fuse to form multinucleated myotubes, which becomecontractile.

As used herein, the term “xenograft” or “xenotransplant” refers to atransplanted cell, tissue, or organ derived from an animal of adifferent species. By way of an example, a graft from a mouse to a humanis a xenograft.

As used herein, the term “xenotransplantation” refers to the process oftransplantation of living cells, tissues or organs from one species toanother, such as from mice to humans.

As used herein, the term “disease model” as used herein refers to theuse of non-human animal models to obtain new information about humanmuscular diseases. In some embodiments, a population of cells fromdystrophic patients, generated as the LHCN cells were generated, andinjected into mice following the methods as disclosed herein, can beused in disease modeling experiments.

As used herein, the term “drug screening” as used herein refers to theuse of cells and tissues in the laboratory to identify drugs with aspecific function. In some embodiments, the present invention provides asubject for drug screening to identify compounds or drugs useful astherapies for diseases or illnesses (e.g. human muscle diseases orillnesses).

As used herein, the term “engrafting” or “engraftment” is used herein torefer to the ability of hMPCs or LHCN cells, provided bytransplantation, to repopulate a tissue. The term encompasses all eventssurrounding or leading up to engraftment, such as tissue homing ofcells, colonization of cells within the tissue of interest, and growthand differentiation of these cells into mature tissue. The engraftmentefficiency or rate of engraftment can be evaluated or quantified usingany experimentally acceptable parameter as known to those of skill inthe art and can include cellular number and size. If the engrafted cellsare engineered to express biologically active compounds, engraftment canalso be quantified by the effects of these compounds, e.g., on thesurvival of the recipient. In one embodiment, engraftment is determinedby measuring bioluminescence during a post-transplant period.

As used herein, the terms “subject” and “individual” are usedinterchangeably herein, and refer to an immunodeficient animal (e.g.,either genetically or due to administration of irradiation such asX-rays or administration of immunosuppressive drugs), such as a mouse orhuman, to whom hMPCs or LHCN cells as disclosed herein can be implanted.The term “subject” also encompasses any vertebrate including but notlimited to mammals, (e.g., humans, mice, rats), reptiles, amphibians andfish. However, advantageously, the subject is a mammal such as a mouseor human, or other mammal such as a domesticated mammal, e.g. dog, cat,horse, and the like, or production mammal, e.g. cow, sheep, pig, and thelike are also encompassed in the term subject.

The terms “non-human animals” and “non-human mammals” are usedinterchangeably herein, and include mammals such as, mice, rats,rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.

The term “tissue” refers to a group or layer of similarly specializedcells which together perform certain special functions. The term“tissue-specific” refers to a source or defining characteristic of cellsfrom a specific tissue.

The terms, “enhance,” “enhancing,” and “enhanced” as used herein referto activities whose effects are greater than that which is observed in acontrol or an untreated group or subject. Enhanced activity may bemeasured in vitro, in vivo, or in cell culture studies.

The terms “growth,” grow,” “grown,” or “growing” as used herein, meanthe growth of tissue, including but not limited to one or more tissues,limbs or organs, following an injury of the tissue resulting from adiseases, disorder, trauma or other condition and includes but is notlimited to regeneration as described herein below.

The terms “injury of a tissue” and “tissue injury” as used herein, meandamage of a tissue that disrupts its physical structure resulting in theimpairment of its function.

As used herein, the terms “injury of a limb” and “limb injury” meandamage of a limb such as, for non-limiting examples, a finger, arm orfoot, that involves a trauma to any or all of the tissues included inthe limb.

As used herein, the terms “regenerate,” “regenerating,” or“regeneration” as used herein mean the restoration of a tissue,including but not limited to one or more tissues, limbs or organs, toits original state following an injury of the tissue resulting from adisease, disorder, trauma or other condition.

As used herein, the terms “stimulate,” “stimulating,” and “stimulated”refer to an activities whose effects are greater than that which isobserved in a control or an untreated group. Stimulatory effects may bemeasured in vitro, in vivo or in cell culture studies.

As used herein, the term “therapeutic amount” or “therapeuticallyeffective amount” means an amount that achieves the intended therapeuticeffect of enhancing or stimulating regeneration of a tissue, includingbut not limited to one or more tissues, limbs or organs, in a subject.The full therapeutic effect does not necessarily occur by administrationof one dose and may occur only after administration of a series ofdoses. Thus, a therapeutically effective amount may be administered inone or more administrations per day for successive days.

As used herein, the term “treating” means, means taking steps to obtainbeneficial or desired results, including clinical results, such asalleviating or ameliorating one or more symptoms of a disease;diminishing the extent of disease; delaying or slowing diseaseprogression; ameliorating and palliating or stabilizing a metric(statistic) of disease. “Treatment” refers to the steps taken.

2. OVERVIEW

Studies of the pathogenic mechanism underlying human myopathies andmuscular dystrophies often require animal models, but models of somehuman diseases are not yet available, specifically for human diseasessuch as FSHD, the third most common form of muscular dystrophy inadults. Applicant discovered methods to promote the engraftment anddevelopment of myogenic cells from individuals with such diseases intomature muscle tissue in mice. Ultimately these methods and non-humanmodels provide a useful tool for testing therapeutic drugs and othertherapies.

FIG. 11 is a flow chart, 1100, that illustrates at a high level anexample method for generating mature human muscle fibers in mice,according to an embodiment. Although steps are depicted in FIG. 11, asintegral steps in a particular order for purposes of illustration, inother embodiments, one or more steps, or portions thereof, are performedin a different order, or overlapping in time, in series or in parallel,or are omitted, or one or more additional steps are added, or the methodis changed in some combination of ways.

In step 1101, a subject is identified as immunocompromised and selected,such as a immunocompromised NRG 8-week-old mouse, suitable forxenografting because it cannot reject transplanted myogenic cells. NRGmice are also known as NOD-Rag immunodeficient mice or strainNOD.Cg-Rag1tm1MomII2rgtm1Wjl/SzJ.

In step 1103, the left limb of the subject is subjected to a single,localized dose of X-irradiation or other treatment to suppress theregenerative potential of the muscle in the subject (i.e., drugs,genetic manipulation) which has been shown to suppress >90% of satellitecell activation. The radiation beam was focused on the lower hindlimb.

In step 1105, a toxin such as cardiotoxin (CTX), BaCl₂, or notexin wasprepared and injected into three sites along the length of the muscle(e.g., TA muscle) to selectively induce degeneration of endogenousmyofibers without affecting the blood vessels or muscle innervation.

In step 1107, LHCN cells, or hMPCs of different origins or hMPCsimmortalized from satellite cells from a biopsy of the pectoralis majormuscle of a 41-year-old male Caucasian heart-transplant donor, werefurther transduced with retrovirus to express luciferase constitutively.A single injection was performed into the muscle (e.g., TA compartment)along the bone, during which the needle was withdrawn slowly, to promotebroad distribution of the injected cells. Bioluminescence imaging may beperformed to monitor the survival and development of the grafted cellsnon-invasively by injecting a solution of D-luciferin IP into thesubject and allowing it to distribute through the body. Imaging wasperformed.

In step 1109, intermittent neuromuscular electrical stimulation wasperformed to determine if it enhanced engraftment of the LHCN cells orhMPCs. The ankle dorsiflexors of the subject (i.e., mice) werestimulated by electrical stimulation through the intact skin over thecommon peroneal nerve. Muscle in the engrafted region may be removed andfrozen cross sections prepared. Immunolabeling can be performed on eachmuscle (e.g., TA muscle) sample using mouse monoclonal antibodiesspecific for human β-spectrin and human lamin A/C. Imaging can beperformed.

3. EXAMPLE EMBODIMENTS Example 1: Methods and Materials Animals

Eight-week-old NOD-Rag1^(null)IL2_(ry) ^(null) (NOD-Rag) immunodeficientmale mice (strain NOD.Cg-Rag1tm1Mom II2rgtm1Wjl/SzJ; JacksonLaboratories™, Bar Harbor, Me.) were used. These non-obese diabetic(NOD)-congenic mice harbor the Rag1^(null) mutation on chromosome 2 andthe IL2ry^(null) mutation on the X-chromosome, which results in theabsence of T, B and NK cells. This strain of mice is suitable for musclexenografting as they tolerate high levels of irradiation-conditioning,avoid rejection of the transplanted human myoblasts, and hence allowefficient engraftment, differentiation and maturation of implantedmyogenic cells (Silva-Barbosa et al., 2005). All protocols were approvedby the Institutional Animal Care and Use Committee of the University ofMaryland, Baltimore.

X-Ray Irradiation

The hindlimbs of young adult mice (8 weeks old) were subjected to asingle, localized dose of X-radiation (25 Gy at 2.5 Gy/min) as described(Lovering et al., 2007). This dose has been previously shown tosuppress >90% of satellite cell activation following CTX treatment(Roche at al., 2010). Other penetrating forms of ionizing irradiation orother treatments that effectively suppress muscle regeneration may beused. Briefly, mice were anesthetized by an intraperitoneal injection ofa 2:1 mixture of 80 mg/kg ketamine (Butler Schein Animal Health, Dublin,Ohio) and 7 mg/kg xylazine (Akom, Decatur, Ill.) and placed within alead box. A person of ordinary skill in the art may use otheranesthetics if determined to be appropriated and included but are notlimited to isofluorane and 2-2-2-Tribromoethanol. The left hindlimb wasexposed through a hole in the box for X-ray irradiation at a single doseof 25 Gy at 2.5 Gy/min. The hindlimb was secured with adhesive tape. Theionizing irradiation was delivered with a Pantak-Seifert™ 250 KpVX-Irradiator (bipolar series model HF 320, East Haven, Conn.). Theradiation beam was focused onto the lower hindlimb while the rest of thebody was protected by the lead shielding. Ion chamber dosimetry (PTW™model 1006, Freiburg, Germany) was performed outside the collimator toensure delivery of the exact dosage to the hindlimb, as well as insidethe collimator (lead shielding), to monitor backscatter of radiation.

Cardiotoxin (CTX)

Mice were maintained under continuous anesthesia with 2%-2.5%isoflurane. A solution of 0.3 mg/ml cardiotoxin (CTS; Naja mossambica;Sigma™, St. Louis, Mo.) was prepared in sterile phosphate saline buffer(1×PBS, 0.02% sodium azide) and filtered through a sterile 0.2 μm filter(PALL Corporation™; Port Washington, N.Y.). It is possible to use othertoxins such as BaCl₂ and Notexin if preferred. CTX (0.3 mg/ml) wasinjected into three sites along the length of the TA muscle to allowwide distribution of the myotoxin, using a 300 μl 29-gauge tuberculinsyringe (Terumo™, Elktor, Md.) to give a final dose of 20 μg/10 gm bodyweight. CTX injection induces selective degeneration of endogenousmyofibers without affecting the blood vessels or muscle innervation(Couteaux and Mira, 1985).

Human Myoblast Preparation and Transplantation

LHCN cells, hMPCs immortalized from satellite cells from a biopsy of thepectoralis major muscle of a 41-year-old male Caucasian heart-transplantdonor, have been previously described (Zhou et al., 2007). This uniqueimmortalized cell line was further transduced with retrovirus to expressluciferase constitutively, to facilitate in vivo imaging of the graftedmyoblasts.

Cells were grown in medium composed of four parts Dulbecco's modifiedEagle's medium (Gibco™, Life Technologies, Grand Island, N.Y.), to onepart medium 199 (Gibco™ Life Technologies™, Grand Island, N.Y.), andsupplemented with 15% HyClone™ USDA tested fetal bovine serum (ThermoScientific™, Rockford, Ill.), 0.02 M HEPES (Sigma-Aldrich™, St. Louis,Mo.), 0.03 μg/ml zinc sulfate (ZnSO₄) (Fisher Scientific™, Pittsburgh,Pa.), 1.4 μg/ml vitamin B12 (Sigma-Aldrich™, St. Louis, Mo.), 0.055μg/ml dexamethasone (Sigma-Aldrich™, St. Louis, Mo.), 2.5 ng/ml humanhepatocyte growth factor (HGF) (EMD Millipore™, Billerica, Mass.), and10 ng/ml basic fibroblast growth factor (bFGF) (BioPioneer™, San Diego,Calif.) (Zhu et al., 2007). Cells were allowed to proliferate on dishescoated with 0.1% pigskin gelatin (Sigma-Aldrich™, St. Louis, Mo.) untilthey reached 70%-80% confluency. Cells were maintained at 37° C. in 10%CO₂ atmosphere. Myogenic purity of cell cultures was determinedfollowing anti-desmin immunofluorescence staining (diluted 1/100; ThermoScientific™ Rockford, Ill.), as desmin protein is expressed in musclecells and not fibroblasts. Each cell preparation used fortransplantation reached 80%-85% myogenic purity.

Prior to injection, cells were released form the substrate by briefdigestion with trypsin, diluted in an equal volume of growth media andcentrifuged at 1×10³ rpm for 3 min (IEC Centra™ CL2 centrifuge). Cellswere suspended in 50 μl growth media.

Two concentrations of LHCN cells were injected: 5×10⁵ (Group 1, n=18) or2×10⁶ (Group 2, n=21). Additionally some of the mice from Group 2 weresubjected to intermittent neuromuscular electrical stimulation protocol(iNMES) to see if this increased the efficiency of engraftment (Group 3,n=16). Aliquots of cell suspensions containing human immortalized LHCNmyoblast cells (5×10⁵ Group 1) or (2×10⁶ Group 2) were injected into theTA muscle compartment along the bone of NOD-Rag mice followingX-irradiation and CTX muscle injury. The needle was withdrawn slowlyfrom the knee towards the ankle, to promote broad distribution of theinjected cells. A third experimental group was injected with 2×106 cellsand subjected to neuromuscular electrical stimulation, as describedbelow (Group 3).

Bioluminescence Imaging (BLI)

BLI was performed on a subset of animals using the Xenogen™ IVIS® 200system (Caliper Life Sciences™, Hopkinton, Mass.) to monitor thesurvival and development of the engrafted LHCN cells non-invasively.Mice were anesthetized with 2%-2.5% inhaled isoflurane. A solution ofD-Luciferin (40 mg/kg) (Caliper Life Sciences™, Hopkinton, Mass.) wasprepared in sterile 1×PBS and sterilized by filtration through a 0.2 μmfilter (PALL Corporation™). Mice were injected intraperitoneally withthe solution of D-luciferin at a dose of 150 mg/kg. Mice were returnedto their cages for 5 min to allow luciferase biodistribution.Anesthetized mice were placed in a light-tight chamber and the lightemitted from the LHCN-luciferase-expressing myoblasts through the TAgrafted tissue was detected with a cooled charge coupled device camera.Imaging was performed 15 minutes after injection, when the luciferaseactivity reached its peak. A sequence of 12 scans with 5 min intervalswere acquired (a total of 120 min), to determine the peak kinetic time.At regular intervals thereafter, usually on a weekly or biweekly basis,for 4 to 7 weeks, imaging was performed to assess the rate of loss oftransplanted cells over time. Following the 4-week period aftertransplantation, animal images were acquired based on the determinedpeak kinetic value.

Regions of interest (ROI) encompassing the injected area of the TAtissue were selected and the luciferase-mediated light intensity wasquantified using the LIVING IMAGE® 4.3.1 software (Caliper LifeSciences™, Hopkinton, Mass.) and expressed as total counts of photonsper second (photons/sec; total flux). The bioluminescence image (pseudocolor image) was overlaid on a photographic image, with light intensityrepresented with a heat map (blue indicates the least intense and redmost). The majority of the animals were scanned on day 0 (immediatelyafter injection of LCHN myoblasts) and day 30 post transplantation andmany were scanned at intermediate times.

Neuromuscular Electrical Stimulation (NMES)

NMES was performed to determine if it enhanced engraftment of LHCNcells. Previous studies have demonstrated improvement in myogenicpotential of muscle precursor cells (Serena et al., 2008), muscleangiogenesis and muscle force (Ambrosio et al., 2012) in both in vitroand in vivo assays, respectively. Mice were anesthetized with 2%-2.5%isoflurane. The ankle dorsiflexors were stimulated by electrical pulsingthrough the intact skin over the common peroneal nerve over the head ofthe fibula. Monophasic square wave pulses of 0.1 ms duration weredelivered to the stimulation electrode by an S48 Stimulator (GrassInstruments™ Warwick, R.I.). A stimulation isolation unit (model PSIU6;Grass Instruments™, Warwick, R.I.) was used to minimize artifact and toensure that the peak current delivered was no greater than 15 mA. Eachcontraction was for 500 ms (150 Hz pulse frequency), followed by a 500ms rest. A rest time of 2 min was followed between the sets of 10contractions to minimize the effect of fatigue. NMES training wasrepeated four times (a total of 40 contractions), at a frequency ofthree times a week over a period of 4 weeks.

In Vivo Assessment of Contractile Function

The maximal torque generated by the ankle dorsiflexors in treated andcontrol hindlimbs during tetanic stimulation of the peroneal nerve wasmeasured in vivo (n=4 animals tested each time), as previously reported(Lovering et al., 2007; Roche et al., 2008; Roche et al., 2010).Briefly, mice were anesthetized with isoflurane (2%-2.5%) and the limbwas stabilized onto a rig by a 27 G needle placed transosseously throughthe head of the tibia. The foot was further placed onto a torque sensorpedal and stabilized with adhesive tape. The ankle dorsiflexors wereengaged by stimulating the common peroneal nerve through the skin withan electrode using monophasic square pulses, 0.1 ms in duration,delivered by an S48 Stimulator (Grass Instruments™, Warwick, R.I.).Pulse amplitude was adjusted to give maximal twitch tension, after whichthe optimal position of the ankle was determined by giving twitches(single stimuli) at different lengths of the dorsiflexors.

At resting length, the frequency of pulses in a 300 ms pulse-train wereprogressively increased until a maximal fused tetany was obtained;usually 100 HzA stimulation isolation unit (model PSIU6; GrassInstruments™, Warwick, R.I.) was used between the stimulator andelectrode to minimize artifact and to ensure that the peak currentdelivered is no greater than 15 mA. Three separate twitches and tetaniccontractions were recorded and saved for further analysis. Signals fromthe torque sensor were amplified (inline amplifier model DV-05,Sensotec; Columbus, Ohio) and sent to a computer via a 12-bit analog todigital board (LabPC-1200, National Instruments™; Austin, Tex.). Datawere recorded using custom written software (Labview 4.1, NationalInstruments; Austin, Tex.). To assess functional recovery, the treatedTA muscle was compared with control TA of the same animal. Contractileforce was measured 4 weeks after transplantation.

Contractile force was measured similarly, with the followingmodifications. The skin on the limb to be assayed was peeled back toexpose the lower half of the TA muscle. The TA tendon is lassoed with #4braided silk thread. The remainder of the TA muscle is freed fromsurrounding tissue and tied via the thread to the foot plate, which isconnected to a force transducer. The Extensor digitorum longus muscle iscut, and the isolated TA is then adjusted on the footplate to a restingtension of −0.6 N/mm. Electrical stimulation is initiated fromfrequencies of 1-100 Hz and the tension is measured. Mineral oil isapplied to the muscle surface, as needed, to prevent dehydration. TAmuscle can be collected after this procedure for morphological andbiochemical studies.

Histology and Immunofluorescence Labeling

Mice were euthanized by cervical dislocation. The TA muscle in theengrafted region was removed, weighed and embedded with O.C.T. (TissueTek™; Torrance, Calif.), snap frozen in liquid nitrogen and stored at−80° C. Cryosections (10-15 μm thick) were cut and mounted on glassSuperfrost microslides (VWR™, Radnor, Pa.). Immunolabeling was performedon unfixed sections from every TA muscle sample, following standardlaboratory methods.

Mouse monoclonal antibodies specific for human β-spectrin (1:100:Leica™, Buffalo Grove, Ill.) and human lamin A/C (1:200: Leica™, BuffaloGrove, Ill.) were used to label the sarcolemma and nuclear lamina,respectively of cells of human origin. To evaluate the internalorganization of the newly formed human muscle fibers, sections were alsostained with rabbit polyclonal anti-desmin (1:200: Thermo Scientific™,Rockford, Ill.). To detect evidence of neuromuscular junction formationin the nascent myofibers, α-bungarotoxin conjugated with Alexa Fluor®594 (Molecular Probes™; Life Technologies™, Grand Island, N.Y.) wasused. (1:200: BIOSS). Presynaptic terminals were detected withantibodies to the SV2 antigen. Sections that were acetone-fixed andstained with a rabbit polyclonal antibody to collagens VIII (1:50; VWR,Radnor, Pa.) were used to detect matrix collagens.

To reduce non-specific staining due to the reaction of primary mousemonoclonal antibodies with the endogenous mouse tissue'simmunoglobulins, sections were treated with reagents the Mouse-on-Mouse(M.O.M) kit from Vector Laboratories™ (Burlington, Calif.). Briefly,sections were incubated for 1 hour with M.O.M. blocking reagent andfurther with M.O.M. diluent protein concentrate solution at roomtemperature (RT) for 10 min. After three washes in 1×PBS-BSA (1 mg/ml)for 5 min each, sections were incubated overnight at 4° C. with primaryantibodies diluted in the solution of protein concentrate. After threewashes in 1×PBS-BSA (1 mg/ml) for 5 min each, primary mouse and rabbitantibodies were visualized with Alexa™ fluor 488 donkey anti-mouse IgG(H+L) and Alexa™ fluor 568 donkey anti-rabbit IgG (H+L) 568,respectively, (1:200: Molecular Probes™; Life Technologies™, GrandIsland, N.Y.) following 1-2 hours incubation at RT. Sections were washedthree times in 1×PBS supplemented with 1 mg/ml BSA for 5 min each, andmounted in Vectashield™ mounting medium with DAPI(4′,6-Diamidino-2-Phenylindole, Dihydrochloride) (Vector Laboratories™,Burlingame, Calif.).

Sections were viewed using a Zeiss™ LSMS DUO confocal microscope, with40× or 63× objectives (Carl Zeiss™, Germany). Images were digitalizedusing LSM Image browser analysis system. Myofibers of human origin,identified by peripheral labeling with antibodies against humanβ-spectrin were counted. The size of human myofibers was determined bymeasuring minimum Feret's diameter (Briguet et al., 2004). Based ontheir measured sizes, myofibers were divided into three groups (1-9 μm,10-19 μm and >20 um). For each TA sample, the percent of myofiberswithin each group was calculated and compared between the groups.Distances from the largest human myofibers to each of its neighboringmyofibers were measured. Additional morphometric assessment of theengrafted human myofibers was performed using LSM Image browser analysissystem. These measurements included the number of nuclei positive forhuman lamin A/C (Riederer et al., 2012; Skuk et al., 2010; Negroni etal., 2009; Silva-Barbosa et al., 2008), the number of murine myonucleiin myofibers of human origin, identified as myonuclei labeled by DAPIbut not by antibodies to human lamin A/C, and the number of centrallynucleated myofibers.

Statistical Analysis

To compare the total count of donor-derived myofibers between theexperimental groups, data were analyzed using one-way analysis ofvariance (ANOVA). The sizes of the engrafted human myofibers across theexperimental groups were analyzed with the chi-square test and theintermyofiber distance between the largest engrafted myofibers and theirclosest neighboring myofibers was evaluated with Fisher exact tests.Data are presented as the mean±SD or ±SEM. A P value of <0.05 wasconsidered significant.

Example 2: In Vivo Bioluminescence Imaging Reveals Acute Donor Cell Losswithin the First Week Following LHCN Transplantation

BLI was performed to evaluate qualitatively the engraftment efficiencyand quantify the survival of engrafted human myogenic precursor cells(hMPCs) into the TA compartment of the mouse hindlimb. Treated TA muscleprior to LHCN injection is significantly reduced in size (not shown).X-irradiation and cardiotoxin treatment therefore effectively eliminatedthe mouse TA muscle and prevented it from regenerating. Serial images ofthe mice were performed on Day 0 after the injection of the LHCNs, andthen typically at 1, 2, and 4 weeks post-transplantation. Some mice werealso examined at 7 weeks post-engraftment.

Quantitative analysis of the bioluminescence signal was detected as afunction of time after LHCN transplantation. Luminometry of engraftedmice is illustrated in FIG. 1A-D. FIG. 1A-1D illustrate bioluminescentimages of mice, 100, at time periods including day 0, 102, and day 7,104, day 28, 106 and day 49, 108, after injection of LHCN cells. Signalsdetected in the TA compartment are indicated in pseudocolor, 110.

Injection of 5×10⁵ or 2×10⁶ LHCN human myoblasts into the X-irradiatedand cardiotoxin (CTX) pre-treated TA muscles (Groups 1 and 2,respectively) resulted in a robust, though variable, bioluminescencesignal at day 0 (not shown). High levels of bioluminescence were alsodetected for Group 3 (injection of 2×10⁶ LHCN myoblasts followed by NMEStraining).

Luminometric measurements of LHCN survival are shown in FIG. 2 In FIG.2, the graph 200, having an x-axis, 202, and a y-axis, 204, shows thequantitative analysis of bioluminescence as a function of time followingtransplantation, reported as a percentage of the signal on day 0 forGroup 1 mice, 206, Group 2 mice, 208, and Group 3 mice, 210. Within thefirst week post-transplantation, ˜10% of the original signal originatingin the injected LHCN remained for all three groups studied. Thisindicates that the majority of the injected myoblasts in all 3 groupsfailed to survive. At 7 weeks post LHCN transplantation only ˜5% of thebioluminescence signal seen after the initial injection of LHCN cellswas detected in the TA compartments. It should be noted that acombination of increased number of LHCN cells (2×10⁶) and neuromuscularelectrical stimulation (NMES) training at day 7 post injection (Group3), resulted in no significant improvement of the bioluminescencesignal.

Taken together, these findings indicate long-term survival of some theengrafted LHCN cells, however, with a small percent of engrafted cells(<7%) retained in the mouse TA compartments at 7-8 weeks posttransplantation.

Example 3: Assessment of Functional Recovery of the LHCN-Transplanted TAMuscles

Torque and force measurements were performed at four weeks after humanmyoblast transplantation to investigate the ability of the grafts formedby the injected LHCN cells to contract LHCN-transplanted TA musclesfollowing injection of 2×10⁶ myoblasts with (Group 3) or without NMEStreatment (Group 2). The values for the two groups were notsignificantly different from each other (not shown). They were, however,significantly weaker than TA compartments in mice that were notX-irradiated, and therefore recovered much of their contractile activityfollowing treatment with CTX (not shown).

Without being bound by theory, it is suggested that mouse TA xenografts,independently of the original amount of LHCN cells injected, generatedsignificant but variable levels of contractile force which made itdifficult to measure their contractile function in a statisticallyreliable manner. As a result of this variability, it is not possible todetermine if NMES provided significant beneficial effects on the abilityof the transplanted LHCN cells to generate contractile force.

Example 4: Immunohistological Analysis Revealed LHCN MyoblastEngraftment and Maturation into Myofibers

Engrafted TA muscle tissues were collected at 4 weeks after injectingthe LHCN cells and characterized. The mass of the engrafted muscle wascompared with that of the control mouse TA muscle. Morphologicalassessment of the LHCN-transplanted TAs preconditioned withX-irradiation and CTX varied in size from 4 to 14 mg in mass (9.9+/−6.1mg for Group 1; 9.1+/−2.7 mg for Group 2; 12.5+/−11.6 for Group 3;mean+/−SD). These differences were not statistically significant. Thevariability is consistent with the variability in our measurements ofcontractile torque and force, however. By contrast, murine TA muscleshave a mass of 31-51 mg (43.2+/−2.8 mg, mean+/−SD).

Characterization of Myofibers of Human Origin

Frozen cross sections of the human muscle grafts from Groups 1, 2, and 3were prepared. Monoclonal antibodies specific for human β-spectrin wereused to label the sarcolemma of the myofibers in the engrafted region todetermine if they developed at least in part by the fusion of LHCNcells. FIG. 3A-3B show photographs, 300, of myofibers that are at leastpartially of human origin in mice that were either treated, 302, (Group3, FIG. 3 A), or not treated, 304 (Group 2, FIG. 3B) with NMES. At 4weeks post-transplantation, significant numbers of myofibers of humanorigin were found in Groups 2 and 3, injected with 2×10⁶ LHCN cells, butnot in Group 1, injected with 5×10⁵ LHCN cells (not shown; but see FIG.4). The results suggest that NMES treatment for 4 weeks followinginjection of 2×10⁶ LHCN cells (Group 3) results in grafts that containmore human muscle fibers that were larger in size and more closelypacked together than grafts that formed in mice that were not subjectedto NMES (Group 2). These qualitative conclusions were quantitated below.

FIG. 4 is a graph, 400, with an x-axis, 402, and a y-axis, 404, thatillustrates the number of fibers that labeled with antibodies to humanβ-spectrin that we counted in each graft. The results show thatinjection of 5×10⁵ LHCN cells, 406, yielded only one mouse in which asignificant number of myofibers of human origin were detected, with theremaining 13 mice showing no such myofibers. Injection of 2×10⁶ LHCNcells yielded many more fibers of human origin, however. In Group 2, 9of 15 mice had significant numbers of myofibers that labeled withantibodies to human β-spectrin, 408, whereas in Group 3, 11 of 14 micehad fibers of human origin, 410. Comparisons of all 3 groups showed thatthe differences in the numbers of mice containing grafts with fibers ofhuman origin, and the numbers of fibers in these grafts were bothstatistically significant. In particular, Group 3 had a mean±SD numberof fibers of 82.2±79.2, compared to Group 2, with 30.9±46.3 (p<0.05).The largest number of human myofibers in LHCN-generated grafts is 226,or approximately 10% of the number of myofibers in a normal mouse TAmuscle (Sharp et al., 2011). These results confirm that injection of2×10⁶ LHCN cells, but not 5×10⁵ LHCN cells, reliably yields graftscontaining myofibers of human origin, and that NMES further enhances theformation of such grafts and increases the number of human myofibers inthem.

FIG. 5 is a graph, 500, with an x-axis, 502, and a y-axis, 504, thatillustrates the differences the sizes of the myofibers of human originin the grafts. In the sole graft containing human myofibers in Group 1,injected with 5×10⁵ LHCN cells, almost 70% of the myofibers were 9 μm orless in diameter, 506, and the largest myofibers did not exceed 19 μm indiameter, 508. By contrast, more than 10% of the myofibers of humanorigin in Groups 2 and 3 were 20 μm or more in diameter, 510, andapproximately half (Group 2) or 70% (Group 3) of the fibers were 10 μmor more in diameter, 508, 510. Although the mean values measured forminimal Feret's diameter in Group 2, 12.4±7.06 μm, and in Group 3,13.05±6.3 μm, were very similar, there was a significant increase in thenumber of larger myofibers in size in Group 3 over Group 2 (p<0.00001).These results confirm that injection of 2×10⁶ LHCN cells, but not 5×10⁵LHCN cells, reliably yields grafts containing large myofibers of humanorigin, and that NMES increases the number of large human myofibers inthe grafts.

FIG. 6, is a graph, 600, having an x-axis, 602, and a y-axis, 604, thatshows measurements of the distances between the largest human myofibersin the grafts of Group 2 and Group 3 and each of their nearestneighbors. Distances were measured as <2 μm, 606, 2 to 5 μm, 608, and >5μm, 610. In Group 2, the highest percentage of myofibers (60%) wasgreater than 5 μm away from the nearest myofiber. Conversely, in Group 3only about 35% of myofibers were >5 μm. Similarly, in Group 2, only ˜10%of the myofibers of human origin were <2 μm from their nearestneighboring fibers, whereas in Group 3 this value was ˜35%. Overall, theinterfiber distances in Group 3, 3.8±3.1 μm, were significantly lessthan those in Group 2, 7.0±5.6 μm (p<0.0001). Although the intermyofiberdistances in Group 3 were greater than in healthy skeletal muscle(typically <1 μm, these results confirm that the grafts formed by 2×10⁶LHCN cells followed by NMES (Group 3) were more tightly organized thanthose formed by the injection of the same number of cells but withoutNMES treatment (Group 2).

Presence of Collagen in Grafts

The connective tissue formed in the engrafted regions was also examined.Due to variability in the overall morphology of the LHCN-transplanted TAmuscles, quantification of the extent of collagenous tissue in theengrafted regions was not possible. Therefore, only qualitativeassessment of fibrosis was made across experimental groups 2 and 3. FIG.7A-7B illustrate, 700, immunofluorescence staining with antibodies tocollagen I and III and revealed less collagenous materials surroundingthe engrafted human myofibers in Group 3 compared with Group 2 (FIG.7A-7B). The amount of collagen present and labeled by the antibodiesseems to increase as the distances between neighboring fibers increase,consistent with the presence of fibrotic tissue in those gaps. Collagenstaining revealed more extensive fibrosis in the human muscle graftsthat were not (FIG. 7A) subjected to NMES, 702, than in those that were(FIG. 7B) subjected to NMES, 704. The regions between myofibers ingrafts subjected to iNMES contained far less labeling collagen thenuntreated grafts, consistent with the closer packing of myofiberspromoted by NMES. Without being bound by theory, the formation oflarger, more tightly packed fibers in Group 3 is associated with areduction in fibrosis.

Differentiation of Fibers of Human Origin in the Graft

In LHCN-transplanted TAs from Group 3, FIG. 8 is a photograph thatillustrates immunofluorescence staining, 800, of many engrafted humanmyofibers and shows evidence of large, clear cytoplasmic networks ofdesmin in the myoplasm, 802, suggesting an organization of thecontractile apparatus into myofibrils that are surrounded by a networkof intermediate filaments composed at least in part of desmin. This is alate stage in myogenesis, indicating considerable maturity of the tissueformed by the engrafted cells (Capetanaki et al., 2007). Similarreticular networks of desmin were observed in cross-sections of thelargest myofibers in Group 2 (not shown). These results indicate thatthe contractile apparatus in the large fibers of human origin in thegrafts is differentiated, as it is in mature mammalian muscle.

It was confirmed that many of the fibers of human origin in the graftwere terminally differentiated by labeling with DAPI, a nuclear stain.FIG. 9A-9B are photographs, 900, illustrating immunofluorescence imagesof central nuclei in fibers of human origin. Samples were labeled withantibodies to human lamin A/C, 902, as well as with antibodies to human(3-spectrin, 904. In developing or regenerating muscle tissue, nucleiwere frequently found in the middle of the cell, whereas in mature,fully differentiated muscle tissue, nearly all of the myonuclei werelocated peripherally, immediately adjacent to the sarcolemma.Examination of the grafts formed by LHCN cells showed that 82% (121 of149) and 79% (146 of 185) of the myonuclei in the grafts were locatedperipherally in Groups 2 and 3, respectively, and that the remaining˜20% of the myonuclei in both groups were centrally located. Thus, bythis criterion ˜20% of the fibers are in the process of developing orregenerating, while ˜80% were fully differentiated.

These results suggested that the engrafted LHCN cells were capable ofterminally differentiating into mature muscle during the 4-week posttransplantation period, and that, although this ability of the grafts toform myofibers that are >20 μm in diameter is promoted by NMES, NMES isnot strictly required for the differentiation of myofibers in the graft.

The presence of differentiated myofibers of the size found in thexenografts suggested that the fibers formed by LHCN cells wereinnervated. In the absence of innervation, these fibers would atrophysignificantly, and would consequently be greatly reduced in size. Totest for innervation, the grafts of Group 3 were labeled with afluorescent derivative of α-bungarotoxin, to detect the large clustersof acetylcholine receptors in the postsynaptic membrane of theneuromuscular junction. Structures typical of postsynaptic membrane thatlabeled by α-bungarotoxin on muscle fibers of human origin were found,indicating that innervation had occurred within 4 weeks aftertransplantation of LHCN cells. FIG. 10A-10B are photographs, 1000, thatillustrate immunofluorescence staining of labeling of the post-synapticmembrane of the neuromuscular junction, 1002, in grafts treated withiNMES or with antibodies to SV2 antigen to label presynaptic structures,1004. Immunolabeled sections were also examined with antibodies to SV2,a protein found in presynaptic vesicles at motor nerve terminals. SV2was found in structures typical of nerve terminals lying along musclefibers of human origin, consistent with the innervation of these fibersin the xenografts.

It was considered a possibility that, despite the fact the hindlimbs ofmice were irradiated to prevent muscle regeneration following CTXintoxication, the myofibers formed by LHCN cells were hybrids formed bythe fusion of the LHCN cells not only with each other but also withmouse myoblasts. In the absence of a mouse-specific lamin A/C antibody,mouse myonuclei were identified as those DAPI-stained myonuclei withinmyofibers that stained positively for human β-spectrin. To avoid anyambiguity in distinguishing peripheral myonuclei from nuclei of nearbycells, such as those in fibroblasts, capillaries and lymphocytes,analysis was limited to central nuclei, such as those shown in FIG. 9.The numbers of centrally located nuclei of mouse origin was extremelylow in the human myofibers formed by LHCN cells in both in Group 2(1/149, 1.3%), and Group 3 (5/185, 2.7%). These results suggest thatthere was minimal murine contamination of the human myofibers formed byLHCN cell grafts.

Consistent with these results, qualitative assessment of the humanmuscle grafts across the three experimental groups revealed that thevast majority of the mouse myofibers were ablated followingX-irradiation and CTX treatment. Those remaining were typically at theedge of the xenograft containing human muscle tissue, suggesting thatthe latter was free of mouse muscle tissue. Therefore, the combinationof X-irradiation and CTX treatments of the mouse TA muscle not onlyeliminates all or nearly all of the tissue of murine origin, but alsoinhibits the formation of hybrid human-mouse myofibers formed bytransplantation of the LHCN myoblasts.

Repopulation of Engrafted LHCN Cells

Finally, data further suggested that the engrafted LHCN cells may havebegun to repopulate the satellite cell niche in the graft. Donor-derivedmononucleate cells co-expressed Pax7 and human lamin A/C associated withDAPI-positive nuclei at the periphery of myofibers of human origin (datanot shown). This is the expected location of satellite cells. Theability of human muscle precursor cells to repopulate the satellite cellniche following introduction into mouse TAs was previously reported(Skuk et al., 2010).

4. OTHER EMBODIMENTS Methods for Treatment of Muscle Disorders byEnhancing Myoblast Cell Engraftment to Generate Mature Muscle Fibers

In certain embodiments, a subject having a muscle disorder isidentified. The subject may be a mammal such as a mouse or human. If themammal is non-human such as a mouse, the non-human mammal must beimmunocompromised so that it does not reject injection of hMPCs. hMSCsare used to regenerate, repair, or newly generate muscle that has beendamaged through disease or degeneration. In certain embodiments, hMPCsdifferentiate into muscle cells and integrate with the healthy tissue ofthe recipient to replace the function of the dead or damaged cells,thereby repairing and/or regenerating the muscle tissue as a whole.Reduced muscle function can be caused by a number of muscle diseasesincluding, but not limited to those listed in Table 1.

hMPCs are injected into the portion of the limb. In certain embodiments,hMPCs of different origins, distinct from LHCN cells, may be injected.hMPCs may be injected at different times after irradiation andintoxication, at different doses and frequencies, and over a differenttime course, as determined by one of skill in the art. In some aspects,injection can be provided by several routes of administration, includingbut not limited to, intramuscular injection, if the hMPCs are in aliquid suspension preparation or where they are in a biocompatiblemedium which is injectable in liquid form. A conventional intramuscularsyringe can serve as a delivery device and can be used so long as theneedle lumen or bore is of sufficient diameter so as to not damage thehMPCs. The injectable hMPCs can also be administered intravenouslyeither by continuous drip or as a bolus. The nerve of the limb (e.g.,peroneal nerve) is subjected to therapeutic electrical stimulation suchas NMES or other means such as exercise (e.g., running wheel in a cageor on a treadmill) configured to enhance engraftment of the myogenicprecursor cells.

As a representative example of a dose range in a non-human mammal suchas a mouse is a volume of at least about 2×10⁶ hMPCs in 50 μl into theformer TA region. For humans, the dosage would be configured by one ofordinary skill in the art depending on necessity due to the health ofthe subject, extent of muscle disease, muscle injury, and degree of lossof function.

In certain embodiments, the subjects may be rendered incapable ofrejecting the engrafted hMPCs by x-irradiation or by genetic means or bymeans other than genetic means. Such means may include, but are notlimited to, other forms of radiation or treatment with a drug thatsuppresses the immune system. Different doses and time courses forx-irradiation and these other means used to suppress the myogenicpotential of the muscle in the subject and are known in the art.Engrafted limbs may be treated in certain embodiments with drugs,hormones, or other biologically active compounds or materials to promoteengraftment. Such reagents may include, but not be limited to, losartan,IFG1, antibodies to TGF-β proteins, and the region of the ActRIIBreceptor that binds proteins of the TGF-β superfamily.

TABLE 1 Muscle Diseases. Amyotrophic lateral sclerosis (ALS) AndersenTawil Syndrome Becker Muscular Dystrophy (BMD) Becker Myotonia CongenitaBethlem Myopathy Bulbospinal Muscular Atrophy (Spinal Bulbar MuscularAtrophy) Central Core Disease (CCD) Centronuclear Myopathy Charcot MarieTooth Disease (CMT) Congenital Muscular Dystrophy (CMD) CongenitalMyotonic Dystrophy Dejerine-Sottas Disease (DSD) Dermatomyositis (DM)Distal Muscular Dystrophy (DD) Duchenne Muscular Dystrophy (DMD)Dystrophia Myotonica (Myotonic Muscular Dystrophy) Emery-DreifussMuscular Dystrophy (EDMD) Eulenberg Disease (Paramyotonia Congenita)Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) Finnish (Tibial)Distal Myopathy Fukuyama Congenital Muscular Dystrophy Glycogenosis Type4 (fetal only in humans; identified in cats, too) Hauptmann-ThanheuserMD (Emery-Dreifuss Muscular Dystrophy) Hereditary Inclusion-BodyMyositis Hereditary Motor and Sensory Neuropathy (Charcot-Marie-ToothDisease) Hyperthyroid Myopathy Hypothyroid Myopathy Inclusion-BodyMyositis (IBM) Inherited Myopathies Integrin-Deficient CongenitalMuscular Dystrophy Kennedy Disease (Spinal-Bulbar Muscular Atrophy)Kugelberg-Welander Disease (Spinal Muscular Atrophy) Limb-GirdleMuscular Dystrophies (LGMDs) Lou Gehrig's Disease (Amyotrophic LateralSclerosis) Merosin-Deficient Congenital Muscular Dystrophy MetabolicDiseases of Muscle Mitochondrial Myopathy Miyoshi Distal Myopathy MotorNeurone Disease Muscle-Eye-Brain Disease Myasthenia Gravis (MG)Myofibrillar Myopathy Myotonia Congenita (MC) Myotonic MuscularDystrophy (MMD) Nemaline Myopathy Nonaka Distal Myopathy OculopharyngealMuscular Dystrophy (OPMD) Paramyotonia Congenita Periodic ParalysisPeroneal Muscular Atrophy (Charcot-Marie-Tooth Disease) Polymyositis(PM) Pompe Disease (Acid Maltase Deficiency) Progressive ExternalOphthalmoplegia (PEO) Rod Body Disease (Nemaline Myopathy) SpinalMuscular Atrophy (SMA) Spinal-Bulbar Muscular Atrophy (SBMA) SteinertDisease (Myotonic Muscular Dystrophy) Thomsen Disease (MyotoniaCongenita) Ullrich Congenital Muscular Dystrophy Walker-Warburg Syndrome(Congenital Muscular Dystrophy) Welander Distal MyopathyWerdnig-Hoffmann Disease (Spinal Muscular Atrophy)

In some aspects, the present invention generally relates to methods ofgenerating mature human muscle fibers in vivo i.e., in mice, free frommurine myonuclei, and similar in size to mouse muscle fibers. In oneembodiment, the invention provides a method of generating mature humanmuscle fibers in a non-human animal such as a mouse comprising: a)removing the non-human or mouse muscle e.g., TA muscle and preventing itfrom regenerating, b) injecting human myogenic cells into a portion of alimb of the non-human animal or into the volume previously occupied bythe muscle, and c) subjecting the peroneal nerve of the injected limb toelectrical stimulation in vivo. Electrical stimulation has long beenknown to promote muscle differentiation in vitro (e.g., DeDeyne, 2000;Pedroty et al., 2005; Stern-Straeter et al., 2005, Serena et al., 2008)and in vivo (DiStefano et al., 2013; Ambrosio et al., 2012) and NMES hasbeen used therapeutically in man to promote the recovery of skeletalmuscle from injury (Dirks et al., 2014; Bittar and Cliquet, 2010; Kim etal., 2010; Stackhouse et al., 2007). The present invention uses NMES topromote the successful generation of mature human muscle fibers in micein vivo.

NMES involves the use of a device which transmits an electrical impulseto the skin over selected muscle groups by way of electrodes. The NMEScauses muscles to contract as a form of exercise or physical therapy.NMES of healthy muscle is intended to strengthen or maintain muscle massduring or following periods of enforced inactivity, maintain or gainrange of motion, facilitate voluntary muscle control, and temporarilyreduce spasticity. Standard treatment in human can be 3 to 4 sessions aweek for one month when used as adjunctive therapy or muscle retraining.As a means for enhancing engraftment in a nonhuman animal, such as amouse, in certain embodiments, NEMS can be administered intermittentlyfrom one day to about 28 days or longer from the time of injection ofmyogenic cells. More specifically, in about 7 days from injection ofmyogenic cells. This can occur for a period of 4 to 5 weeks. In certainembodiments, NMES can be administered intermittently at 150 Hz for 500msec, repeated 10 times. This can then be repeated 4 times with 2 minuterests, for a total of 40 repetitions, three times per week, for 4-5weeks. One of ordinary skill in the art could change frequency,repetition, and timing to suit experimentation.

The TA muscle may be eliminated by any method known in the art,including, but not limited to, irradiating the hindlimb with X-rays andthen injecting it with a toxin such as cardiotoxin, BaCl₂ or notexin. Asa representative example, cardiotoxin CTX may be injected in a dose of60 μl of a 0.3 mg/ml solution. For example, it is known in the art thatthe neurotoxin from tiger snake venom (notexin, Notechis scutatus) has aLD50 6.4 mg/kg sc mice, minimum dose for myoglobinuria 1.4 mg/kg scmice. Other toxins such as P. australis venom are lethal and can causemyoglobinuria in mice, having a LD50 for 0.25 mg/kg ip mice. SeeLeonardi et al. 1979. One of ordinary skill in the art would know how todetermine the proper dosage for the toxin of choice (i.e., BaCl₂ ornotexin) to induce paralysis. Therapeutic electrical stimulation can beapplied for the first time from between one day and about 14 days fromthe time the myogenic cells are injected. In certain embodimentselectrical stimulation is begun approximately one week after themyogenic cells are injected. The duration and amount of electricalstimulation may range beyond what is specifically used in the workingexamples described herein.

Treatment of the Muscle Disease FSHD

Over the last decade several reports examined the possibility that humanmyoblast therapy is useful for the treatment of genetic muscle diseases.Improved strains of immunodeficient mice have been developed that allowefficient engraftment, differentiation and maturation of human myogeniccells (Huard et al., 1994; Skuk et al., 1999; Pye et al., 2004;Silva-Barbosa et al., 2005; Silva-Barbosa et al., 2008). Earlier studiesof grafts formed by injecting human myogenic precursor cells into miceyielded limited numbers of muscle fibers with human myonuclei (Mouly etal., 2005; Mamchaoui et al., 2011; Reed et al., 2007; Riederer et al.,2012).

Two approaches have been tested to develop grafts of human muscletissues in mice. In one approach, xenografts are created by introducingsmall pieces of mature human muscle myofibers, obtained at biopsy, andsuturing them to muscles in immunodeficient mice (Zhang et al, 2014).Over time, some of these grafts survive and reform mature muscle tissuethat is largely human in origin. But these grafts are fibrotic and canalso contain significant numbers of murine myonuclei. Although the micecarrying these human grafts provide potentially excellent models, itwould be difficult to generate in the numbers needed for therapeutictesting.

As an alternative approach, the ability of human myogenic cells todevelop into mature human muscle tissue in immunodeficient or dystrophicmice has been investigated. (Riederer et al., 2012; Silva-Barbosa etal., 2008; Silva-Barbosa et al., 2005). In these studies, the endogenousmurine muscle is typically eliminated by freezing or injection of atoxin, and, in some cases, regeneration is reduced by priorX-irradiation. Human myoblasts are often tagged with an enzyme such asluciferase (Laumonier et al., 2013; Libani et al., 2011), or afluorescent protein such as green fluorescent protein (GFP) (Benabdallahet al., 2013; Quenneville et al., 2007), to enable them to be tracked byluminometric or fluorescence methods after they are injected into adultmouse skeletal muscle. After injection, mice have been exposed todifferent pharmacological agents, such as Losartan (Fakhfakh et al.,2012b), to alter angiotensin II signaling, and a soluble form of thereceptor for myostatin, ActRIIB-Fc (Fakhfakh et al., 212a), to promotemyogenesis and reduce fibrosis.

These treatments have improved the survival of the engrafted humanmyogenic cells in mice and their differentiation into myofibers.However, the muscles they form are very small and therefore difficult tostudy by physiological morphological and genomic methods. The engraftedmuscle grafts may also contain a large number of murine myonuclei,indicating that they are largely hybrid in nature (Fakhfakh et al.,2012a; Ehrhardt et al., 2007).

In addition, the methods that have been used to suppress murinemyogenesis following injury to murine muscle proved ineffective inpreventing murine muscle from reforming. As the engrafted cells mustcompete with myogenic precursor cells derived from murine satellitecells, they can at best form myofibers of mixed human-mouse origin. Thisis less than optimal for studying the human dystrophic phenotype,considering the likelihood of genetic complementation compensating forthe cause of the myopathy in hybrid fibers.

Studies of the pathogenic mechanisms underlying muscular dystrophiesoften require animal models, but models of some human muscle diseasesare not yet available. Of specific interest in certain embodiments isFSHD. FSHD is one of the most common forms of muscular dystrophy inadults, caused by an epigenetic derepression of the macrosatelliterepeat D4Z4 in the 4q35 region, either by contraction of the repeatarray (FSHD1; Wijmenga et al., 1992; vanDeutekom et al., 1993) or bymutations in modifier genes, such as SMCHD1 (FSHD2) (Lemmers et al.,2012). These genetic and epigenetic changes combined withFSHD-permissive chromosomal alleles (Lemmers et al., 2010) can lead toan ectopic expression of the DUX4 retrogene, a potent germlinetranscription factor, residing in each D4Z4 repeat.

Although accumulating evidence supports the possible pathogenic role ofDUX4 in FSHD development, DUX4 may not be the sole candidate gene. Arecent model proposes that deregulation of a global transcriptionalcascade involving numerous DUX4 target genes, initially triggered byDUX4 activation randomly in rare FSHD myonuclei, could lead to muscleatrophy, inflammation, oxidative stress and defects in differentiationprocess, all key features of FSHD (Tassin et al., 2012). Pathogenesis inFSHD may also be linked to other gene products, including long noncodingRNAs (Cabianca et al., Cell. 2012; PMID: 22541069) and miRNAs (Cheli etal., PLoS One. 2011; PMID: 21695143). Despite extensive study, themolecular mechanisms underlying FSHD are still not fully understood.

As a result, a valid mouse model of the disease was in need ofdevelopment. Without one, studies of the disease in vivo aresignificantly limited. Several laboratories have generated mice bytransgenic or viral expression of DUX4 (Krom et al., 2012; Wallace etal., 2011), or potential downstream mediators of their activity, such asPITX1 (Dixit et al., 2007) and CRYM (Reed et al., 2007; our unpublisheddata). None have so far reproduced the phenotype seen in biopsies ofFSHD patients. Indeed, the likelihood that FSHD is caused by bothgenetic and epigenetic changes (Jones et al., 2012; Lemmers et al.,2012; Lemmers et al., 2010; Rahimov et al., 2012), regulated by modifiergenes that remain to be identified (Jones et al., 2012), casts doubt onthe possibility that a simple transgenic model can be created bymanipulating individual genes.

In certain aspects of the invention, a murine model for FSHD shouldreproduce all the features of FSHD muscle, retaining the morphological,physiological and genomic differences found in fresh biopsies. As themechanisms underlying pathogenesis in FSHD are still unclear, it isnecessary to construct muscle tissue for study in vivo frompatient-derived human myogenic cells, which presumably retain thesedisease-causing changes. Applicant optimized transplantation conditionsfor successful engraftment of a cell line derived from a FSHD patientusing immortalized human myogenic precursor cells and injecting thesemyoblast cells into the tibialis anterior (TA) muscle compartment in theanterior hindlimb of immunodeficient NOD-Rag1^(null)IL2_(ry) ^(null)mice (NOD-Rag).

In one embodiment, hMPCs from a patient with Facioscapulohumeralmuscular dystrophy were introduced into mice, following the methodsdescribed herein, including NMES, muscle fibers of human origindeveloped and expressed several genes typical of FSHD tissue. Inparticular, the fibers generated following engraftment of these cellslabeled with antibodies to human β-spectrin and to human lamin A/C (notshown). Furthermore, when harvested and assayed by quantitative RT-PCRmethods and compared to the grafts formed by LHCN cells, they containedseveral fold higher levels of mRNA encoding DUX4 (not shown), a proteinbelieved to be involved in the pathogenesis of FSHD. Consistent withthis, quantitative RT-PCR methods also showed significant increases inmRNAs encoding at least two gene products that are transcribed when DUX4is elevated (not shown). These results indicate that our methods are notlimited to LHCN cells, that they are generally applicable to hMPCs,including hMPCs derived from subjects with neuromuscular disease, andthat the grafts that they produce retain many of the characteristics ofthe tissue from which the hMPCs originated.

The usefulness of certain embodiments was also tested by creatingxenografts of hMPCs from diseased tissue in mimicking the diseasephenotype in mice. Immortalized hMPCs from a patient with FSHD were usedfor this purpose. These cells, like LHCN cells, had been transduced toexpress firefly luciferase. Using the methods described, it was foundthat FSHD cells engrafted in mice at least as well as in controls. PCRfor DUX4 was performed on mRNA from 2 grafts (G31M3, P19M2), and 2 mouseTA muscles. 17ABic was a positive control for Dux4. hRPL13A was ahuman-specific marker. The grafts formed from FSHD hMPCs express Dux4but the controls do not (data not shown). qPCR for two Dux4 downstreamgenes was performed on mRNA from 1 graft formed by LHCN cells and 2 byFSHD cells. The results show a significant increase (p<0.05) inexpression of TRIM43 and ZSCAN4 in grafts formed by the FSHD hMPCs (datanot shown). Therefore, xenografts formed by FSHD hMPCs preserve thedistinct genetic program observed in the hMPCs in culture and in thebiopsies from which those cells were originally derived. With furtheroptimization, certain embodiments will yield mice carrying mature FSHDmuscles. The mice carrying these grafts should be suitable for testingtherapies to treat FSHD. The most sensitive and reliable assays utilizeqRT-PCR of FSHD-specific biomarkers, which are applicable to Applicant'smouse model. A therapeutic drug for FSHD should bring biomarker levelsto close to controls. Thus, the methods described here should make itpossible to screen compounds and compare therapeutic efficacies ofdifferent drugs on living FSHD muscle tissue in vivo.

In certain embodiments, optimization of these unique xenograftingconditions for successful transplantation of normal immortalized humanmyoblasts in mice will allow future testing of similar immortalized celllines derived from FSHD patients and their first-degree relatives(Mamchaoui et al., 2011; Stadler et al., 2011). It is expected that themature myofibers derived from immortalized FSHD myoblasts will show thephenotype of FSHD muscle, with all the associated genetic, epigenetic,physiological and morphological properties (Geng et al., 2012; Jones etal., 2012; Lassche et al., 2013; Lemmers et al., 2010; Rahimov et al.,2012; Reed et al., 2006), independently of any particular molecularmodel of pathogenesis.

Considering the feasibility of assaying this mature human muscle tissuein mice morphologically and physiologically, and the validity and use ofLHCN immortalized normal cells as a universal human cellular model,other embodiments are directed to the application of such axenotransplant model to not only FSHD, but a wide variety ofneuromuscular disorders.

Methods for Producing a Non-Human Model for Human Muscle Disease

The invention further provides a method for producing a non-human animalthat models a human muscular disease. A non-human animal such as animmunocompromised mouse suitable for xenografting is obtained. Thehindlimb of the non-human animal is irradiated with X-rays. The TAmuscle is extracted and a cardiotoxin or other toxin (e.g., BaCl₂ ornotexin) is injected along the length of the TA muscle to inducedegeneration of myofibers of the TA muscle in the non-human animalthereby and creating a volume or compartment where TA muscle used to belocated. HMPCs or LHCN cells obtained from a human muscular disease aretagged with an enzyme such as luciferase and injected into the non-humanTA muscle compartment to form a graft. The peroneal nerve of theinjected hindlimb is subjected to electrical stimulation. Contractilefunction of myofibers of human origin is then assessed. The graftgenerated by human myogenic precursor cells obtained from a humanmuscular disease may then be compared with cells obtained from anon-diseased muscle and ultimately screened for therapeutic drugs.Non-human animal models generated by this method are also contemplated.

In some embodiments where hMPCs are implanted into an animal subject,the animal can be used as an in vivo humanized model of musculardisease. For example, an animal model which comprises functional,innervated, vascularized human muscle tissue, can be used to screen fortherapeutic agents, viruses or drugs which affect any one, or acombination of viability, functionality, contractibility,differentiation of the human muscle tissue.

Accordingly, one embodiment relates to the use of an in vivo humanizedmodel of muscle disease as an assay, for example to assess drug toxicity(e.g. myotoxicity) on human muscle tissue in vivo (e.g. to identifyagents which increase apoptosis, decrease viability, modulate (e.g.increase or decrease by a statistically significantly amount)contractibility and/or conductivity of muscle tissue). In someembodiments, the drugs and/or compounds can be existing drugs orcompounds, and in other embodiments, the drugs or compounds can be newor modified drugs and compounds.

Any suitable immunodeficient animal can be used for implanting apopulation of hMPC cells to generate an in vivo humanized model ofmuscle disease as disclosed herein, (such as NRG mice, NOD-Rag mice,nude mice, such as SCID mice, or animals rendered immunodeficientchemically or by irradiation). The human muscle tissues can be harvestedafter a period of growth, and assessed as to whether the human muscletissue is still present, viable and functioning normally.

In some embodiments, the LHCN cells or hMPCs of other originadministered to the subject can be transduced with retrovirus to expressa detectable label such as luciferase constitutively, to facilitate invivo imaging after engraftment. Others can express a detectable label(such as green fluorescent protein, or beta-galactosidase);alternatively, they may have been prelabeled (for example, with BrdU or^(3H)thymidine), or they may be detected by virtue of their expressionof a constitutive cell marker (for example, using human-specificantibody). The presence and phenotype of the administered LHCN cells canbe assessed several methods, including but not limited to luminometry,immunohistochemistry, or RT-PCR analysis using primers and hybridizationconditions that cause amplification to be specific for humanpolynucleotides, according to published sequence data.

The effect of an agent administered to an in vivo humanized model ofmuscle disease can be assessed by many methods, including but notlimited to improvement in muscle histology, improvement in contractilefunction, reduction of fibrosis or fatty infiltrates, reduction in theexpression of disease-specific markers, increased expression of musclespecific proteins or mRNAs, or improved recuperation followingintentional injury to the humanized muscle tissue.

In such an embodiment, the in vivo humanized model of muscle disease ofthe invention can be used to screen for agents which alleviate theinjury. In alternative embodiments, the in vivo humanized model ofmuscle disease can be assessed by the degree of muscle recuperation thatensues from inflicting injury to the human muscle tissue. The manner inwhich human muscle tissue responds to an agent, particularly apharmacologic agent, including the timing of responses, is an importantreflection of the physiologic state of the human muscle tissue.

The use of the in vivo humanized model of muscle disease as disclosedherein provides significant advantages over existing methods to assessagents on muscle tissue, because the in vivo humanized model of muscledisease comprises human muscle tissue which is formed from e.g., LHCNsor hMSCs in vivo, and is properly re-vascularized and comprises all thedesired cell types of muscle tissue, including cells of myocytephenotypes, as well as characteristics and properties of functionalmuscle tissue. This is highly advantageous as it provides a model ofhuman muscle tissue in vivo, which is significantly advantageous overexisting muscle function assays which either are assays using humanmuscle tissue in vitro, or are in vivo models using non-human muscletissue.

In some embodiments, an agent administered to an in vivo humanized modelof muscle disease as disclosed herein can be selected from a group of achemical, small molecule, chemical entity, nucleic acid sequences,nucleic acid analogues or protein or polypeptide or analogue of fragmentthereof. In some embodiments, the nucleic acid is DNA or RNA, andnucleic acid analogues, for example can be peptide nucleic acid “PNA,”pseudo-complementary peptide nucleic acids, “pcPNA” and locked nucleicacids, “LNA.” A nucleic acid may be single or double stranded, and canbe selected from a group comprising; nucleic acid encoding a protein ofinterest, oligonucleotides, PNA, etc. Such nucleic acid sequencesinclude, for example, but not limited to, nucleic acid sequence encodingproteins that act as transcriptional repressors, antisense molecules,ribozymes, small inhibitory nucleic acid sequences, for example but notlimited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisenseoligonucleotides etc. A protein and/or peptide agent or fragmentthereof, can be any protein of interest, for example, but not limitedto; mutated proteins; therapeutic proteins; truncated proteins, whereinthe protein is normally absent or expressed at lower levels in the cell.Proteins of interest can be selected from a group comprising; mutatedproteins, genetically engineered proteins, peptides, synthetic peptides,recombinant proteins, chimeric proteins, antibodies, humanized proteins,humanized antibodies, chimeric antibodies, modified proteins andfragments thereof.

In some embodiments, at least one agent is administered to an in vivohumanized model of muscle disease as disclosed herein by any suitablemeans known to one of ordinary skill in the art. In some embodiments,administration occurs more than once, for example at multiple differenttime points. In some embodiments, the administration of an agent to anin vivo humanized model of muscle disease is continuous, for example viameans of an infusion pump or catheter or the like, or via a slow-releaseformulation of the agent. In some embodiments, the agent is administeredlocally to the site of the human muscle tissue in the in vivo humanizedmodel of muscle disease, or alternatively, systemically to the in vivohumanized model of muscle disease.

In some embodiments, an agent is administered to an in vivo humanizedmodel of muscle disease via any or a combination of the followingadministration methods; systemic administration, intravenous,transdermal, intrasynovial, intramuscular, oral administration,parenteral administration, intraarterial administration, intrathecaladministration, intraventricular administration, intraparenchymal,intracranial, intracisternal, intrastriatal, and intranigraladministration, and intracoronary administration.

In some embodiments, the agents are conveniently administered to an invivo humanized model of muscle disease in a pharmacological applicablecarrier, such as solution, or readily soluble form. The agents may beadded in a pump (e.g. flow-through system), as a stream, intermittent orcontinuous, or alternatively, adding a bolus of the compound, singly orincrementally, to an otherwise static solution. In some embodiments,agent formulations do not include additional components, such aspreservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

In some embodiments, an agent may be applied to the media comprising theLHCN cells or hMPCs prior to the implantation into the subject, wherethe agent contacts the LHCN cells and induces its effects.Alternatively, the agent may be intracellular within the LHCN cells as aresult of introduction of the nucleic acid sequence into the cell andits transcription resulting in the production of the nucleic acid and/orprotein agent within the cell.

In some embodiments, an agent also encompasses any action and/or eventto which the in vivo humanized model of muscle disease as disclosedherein is are subjected. As a non-limiting example, an action cancomprise any action that triggers a physiological change in human muscletissue in the in vivo humanized model of neuromuscular disease asdisclosed herein, for example but not limited to; heat-shock, ionizingirradiation, cold-shock, electrical impulse, light and/or wavelengthexposure, UV exposure, pressure, stretching action, increased and/ordecreased oxygen exposure, exposure to reactive oxygen species (ROS),ischemic conditions, fluorescence exposure etc. Environmental stimulialso include intrinsic environmental stimuli defined below. The exposureto agent may be continuous or non-continuous.

Methods for Determining Myotropism of Viruses for Gene Therapy

In other embodiments, the mice carrying the grafts formed by LHCN cellshave potentially broad uses, e.g., determining the myotropism of virusesfor gene therapy, assessing the effects of drugs, antibodies,microorganisms and toxins on mature human muscle, and developing newapproaches to treating a number of human diseases of muscle.Specifically applied to muscular dystrophies such as FSHD, the methodsare significant because they provide an excellent means of generatingmature FSHD muscle tissue in mice, leading to a valuable tool for thestudy of pathology of the disease and its treatment, including thepossible use of autologous myoblast-based therapies.

For purposes of the present invention, gene therapy refers to thetransfer and stable insertion of new genetic information into cells forthe therapeutic treatment of muscle diseases or muscle disorders. Aforeign sequence or gene is transferred into hMPCs that proliferate tospread the new sequence or gene throughout the cell population. Knownmethods of gene transfer include microinjection, electroporation,liposomes, chromosome transfer, transfection techniques,calcium-precipitation transfection techniques, and the like. Forexample, muscular dystrophy may result from a loss of gene function, asa result of a mutation for example, or a gain of gene function, as aresult of an extra copy of a gene, such as three copies of a wild-typegene, or a gene over expressed as a result of a mutation in a promoter,for example. Expression may be altered by activating or deactivatingregulatory elements, such as a promoter. A mutation may be corrected byreplacing the mutated sequence with a wild-type sequence or inserting anantisense sequence to bind to an over expressed sequence or to aregulatory sequence.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of gene therapy, in accordance with this embodiment of theinvention. The technique used should provide for the stable transfer ofthe heterologous gene sequence to the precursor cell, so that theheterologous gene sequence is heritable and expressible by stem cellprogeny, and so that the necessary development and physiologicalfunctions of the recipient cells are not disrupted. Techniques which maybe used include but are not limited to chromosome transfer (e.g., cellfusion, chromosome-mediated gene transfer, micro cell-mediated genetransfer), physical methods (e.g., transfection, spheroplast fusion,microinjection, electroporation, liposome carrier), viral vectortransfer (e.g., recombinant DNA viruses, recombinant RNA viruses) andthe like (described in Cline, M. J., 1985, Pharmac. Ther. 29:69-92,incorporated herein by reference in its entirety). These methods may beextended to precursor cells transfected with plasmid constructs thatencode a secreted protein, allowing the grafts that are generated toprovide systemic benefit to the subject, e.g., replacement of defectiveor absent polypeptide hormone, and serum factors.

In other embodiments, muscle tissue with hereditary muscle disease maybe identified. hMPCs obtained from this muscle tissue may be isolated.It is possible to then insert a gene into these hMPCs, usingconventional molecular biology techniques known to those in the art, toreplace a gene that is mutated or missing, while maintaining exactly thesame genetic background. The hMPCs expressing the healthy gene may beinjected into a subject in need of muscle repair. Ultimately, the musclein which the genetic defect is so corrected can reform.

Pharmaceutical Compositions and Kits Comprising them

In certain embodiments, pharmaceutical compositions comprising cellsand/or different drugs, different hormones or other biologically activecompounds may be used to promote engraftment or to optimize generationof new muscle tissue. Such reagents may include, but are not limited to,losartan, IGF1, antibodies to TFG-β proteins, and the region of theActRIIB receptor that binds proteins of the TGF-β superfamily. In otherembodiments, kits comprising the pharmaceutical composition may preventrejection of the graft. The pharmaceutical composition and kitcomprising it may be useful for repairing wounded or injured muscle, inaddition to diseased muscle. For example, the methods may be applicableto treating subjects with hMPCs derived from their own healthy musclesin order to replace muscle tissue lost to wounds (e.g., injury due tomilitary combat).

In some embodiments, the present invention relates to a pharmaceuticalcomposition and kits comprising them comprising the myoblast cellscapable of forming muscle tissue as disclosed herein. The pharmaceuticalcompositions may further comprise a pharmaceutically acceptable carrier.The myoblast composition can be administered to a subject alone or incombination with other cells, tissue, matrix components, tissuefragments, or growth factors as disclosed herein. In alternativeembodiments, other known growth factors can be administered incombination with the myoblast cells, e.g., resorbable plastic scaffolds,or other additive intended to enhance the delivery, efficacy,tolerability, vascularization, or function of the implanted myoblastcell population.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention.

REFERENCES

All references listed below and throughout the specification are herebyincorporated by reference in their entirety.

-   1. Mamchaoui K, Trollet C, Bigot A, Negroni E, Chaouch S, et al    (2011). Immortalized pathological human myoblasts: towards a    universal tool for the study of neuromuscular disorders. Skeletal    Muscle; 1:34.-   2. Stadler G, Chen J C, Wagner K, Robin J D, Shay J W, Emerson C P    Jr, Wright W E (2011). Establishment of clonal myogenic cell lines    from severely affected dystrophic muscles—CDK4 maintains the    myogenic population. Skelet Muscle; 1:12.-   3. Partridge T A (2013). The mdx mouse model as a surrogate for    Duchene muscular dystrophy. FEBS J; 280:4177-4186.-   4. Kobayashi K, Izawa T, Kuwamura M, Yamate J (2012). Dysferlin and    animal models for dysferlinopathy. J Toxicol Pathol; 25:135-147.-   5. Quattrocelli M, Cassano M, Crippa S, Perini I, Sampaolesi M    (2010). Cell therapy strategies and improvements for muscular    dystrophy. Cell Death Differ; 17:1222-1229.-   6. Vainzof M, Ayub-Guerrieri D, Onofre P C, Martins P C, Lopes V F,    Zilberztajn D, et al (2008). Animal models for genetic neuromuscular    diseases. J Mol Neurosci; 3:241-248.-   7. Zhang Y, King O D, Rahimov F, Jones T I, Ward C W, Kerr J P, et    al (2014). Human skeletal muscle xenograft as a new preclinical    model for muscle disorders. Hum Mol Genet; 23:3180-3188.-   8. Riederer I, Negroni E, Bencze M, Wolff A, Aamiri A, Di Santo J P,    et al (2012). Slowing down differentiation of engrafted human    myoblasts into immunodeficient mice correlates with increased    proliferation and migration. Mol Ther; 20:146-154.-   9. Silva-Barbosa S D, Butler-Browne G S, de Mello W, Riederer I, Di    Santo J P, Savino W, et al (2008). Human myoblast engraftment is    improved in laminin-enriched microenvironment. Transplantation;    85:566-575.-   10. Silva-Barbosa S D, Butler-Browne G S, Di Santo J P, Mouly V    (2005). Comparative analysis of genetically engineered    immunodeficient mouse strains as recipients for human myoblast    transplantation. Cell Transplant; 14:457-467.-   11. Laumonier T, Pradier A, Hoffmeyer P, Kindler V, Menetrey J    (2013). Low molecular weight dextran sulfate binds to human    myoblasts and improves their survival after transplantation in mice.    Cell Transplant; 22:1213-1226.-   12. Libani I V, Lucignani G, Gianelli U, Degrassi A, Russo M, Bosari    S, et al (2012). Labeling protocols for in vivo tracking of human    skeletal muscle cells (HSkMCs) by magnetic resonance and    bioluminescence imaging. Mol Imaging Biol; 14:47-59.-   13. Benabdallah B F, Duval A, Rousseau J, Chapdelaine P, Holmes M C,    Haddad E, et al (2013). Targeted gene addition of microdystrophin in    mice skeletal muscle via human myoblast transplantation. Mol Ther    Nucleic Acids; 2:e68.-   14. Quenneville S P, Chapdelaine P, Skuk D, Paradis M, Goulet M,    Rousseau J, et al (2007). Autologous transplantation of muscle    precursor cells modified with a lentivirus for muscular dystrophy:    human cells and primate models. Mol Ther; 15:431-438.-   15. Fakhfakh R, Lamarre Y, Skuk D, Tremblay J P (2012b). Losartan    enhances the success of myoblast transplantation. Cell Transplant;    21:139-152.-   16. Fakhfakh R, Lee S J, Tremblay J P (2012a). Administration of a    soluble activin type IIB receptor promotes the transplantation of    human myoblasts in dystrophic mice. Cell Transplant; 21:1419-1430.-   17. Ehrhardt J, Brimah K, Adkin C, Partridge T, Morgan J (2007).    Human muscle precursor cells give rise to functional satellite cells    in vivo. Neuromuscul Disord; 17:631-638.-   18. Serena E, Flaibani M, Carnio S, Boldrin L, Vitiello L, De Coppi    P, Elvassore N. Electrophysiologic stimulation improves myogenic    potential of muscle precursor cells grown in a 3D collagen scaffold.    Neurol Res; 30:207-214.-   19. De Deyne P G (2000). Formation of sarcomeres in developing    myotubes: role of mechanical stretch and contractile activation. Am    J Physiol Cell Physio; 279:C1801-1811.-   20. Distefano G, Ferrari R J, Weiss C, Deasy B M, Boninger M L,    Fitzgerald G K, et al (2013). Neuromuscular electrical stimulation    as a method to maximize the beneficial effects of muscle stem cells    transplanted into dystrophic skeletal muscle. PLoS One; 8:e54922.-   21. Ambrosio F, Fitzgerald G K, Ferrari R, Distefano G, Carvell G    (2012). A murine model of muscle training by neuromuscular    electrical stimulation. J Vis Exp; (63):e3914.-   22. Dirks M L, Wall B T, Snijders T, Ottenbros C L, Verdijk L B, and    van Loon L J (2014). Neuromuscular electrical stimulation prevents    muscle disuse atrophy during leg immobilization in humans. Acta    Physiol (Oxf); 210:628-641.-   23. Bittar C K, and Cliquet A Jr (2010). Effects of quadriceps and    anterior tibial muscles electrical stimulation on the feet and    ankles of patients with spinal cord injuries. Spinal Cord;    48(12):881-885.-   24. Kim K M, Croy T, Hertel J, Saliba S (2010). Effects of    neuromuscular electrical stimulation after anterior cruciate    ligament reconstruction on quadriceps strength, function, and    patient-oriented outcomes: a systematic review. J Orthop Sports Phys    Ther; 40:383-391.-   25. Stackhouse S K, Binder-Macleod S A, Stackhouse C A, McCarthy J    J, Prosser L A, Lee S C (2007). Neuromuscular electrical stimulation    versus volitional isometric strength training in children with    spastic diplegic cerebral palsy: a preliminary study. Neurorehabil    Neural Repair; 21:475-485.-   26. Zhu C H, Mouly V, Cooper R N, Mamchaoui K, Bigot A, Shay J W, et    al (2007). Cellular senescence in human myoblasts is overcome by    human telomerase reverse transcriptase and cyclin-dependent kinase    4: consequences in aging muscle and therapeutic strategies for    muscular dystrophies. Aging Cell; 6:515-523.-   27. Ambrosio F, Ferrari R J, Distefano G, Plassmeyer J M, Carvell G    E, Deasy B M, et al (2010). The synergistic effect of treadmill    running on stem-cell transplantation to heal injured skeletal    muscle. Tissue Eng Part A; 16:839-849.-   28. Bouchentouf M, Benabdallah B F, Mills P, Tremblay J P (2006).    Exercise improves the success of myoblast transplantation in mdx    mice. Neuromuscul Disord; 16:518-529.-   29. Pearson T, Shultz L D, Miller D, King M, Laning J, Fodor W, et    al (2008). Non-obese diabetic-recombination activating gene-1    (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r    gamma null) null mice: a radioresistant model for human    lymphohaematopoietic engraftment. Clin Exp Immunol; 154:270-284.-   30. Lovering R M, Roche J A, Bloch R J, De Deyne P G (2007).    Recovery of function in skeletal muscle following 2 different    contraction-induced injuries. Arch Phys Med Rehabil; 88:617-625.-   31. Roche J A, Lovering R M, Roche R, Ru L W, Reed P W, Bloch R J    (2010). Extensive mononuclear infiltration and myogenesis    characterize recovery of dysferlin-null skeletal muscle from    contraction-induced injuries. Am J Physiol Cell Physiol;    298:C298-312.-   32. Harris J B (2003). Myotoxic phospholipases A2 and the    regeneration of skeletal muscles. Toxicon; 42:933-945.-   33. Couteaux R, Mira J C, d'Albis A (1988). Regeneration of muscles    after cardiotoxin injury. I. Cytological aspects. Biol Cell;    62(2):171-182.-   34. Briguet A, Courdier-Fruh I, Foster M, Meier T, Magyar J P    (2004). Histological parameters for the quantitative assessment of    muscular dystrophy in the mdx-mouse. Neuromuscul Disord; 14:675-682.-   35. Riederer I, Negroni E, Bencze M, Wolff A, Aamiri A, Di Santo J    P, et al (2012). Slowing down differentiation of engrafted human    myoblasts into immunodeficient mice correlates with increased    proliferation and migration. Mol Ther; 20:146-154.-   36. Skuk D, Paradis M, Goulet M, Chapdelaine P, Rothstein D M,    Tremblay J P (2010). Intramuscular transplantation of human    postnatal myoblasts generates functional donor-derived satellite    cells. Mol Ther; 18:1689-1697.-   37. Negroni E, Riederer I, Chaouch S, Belicchi M, Razini P, Di Santo    J, et al (2009). In vivo myogenic potential of human CD133+    muscle-derived stem cells: a quantitative study. Mol Ther;    17:1771-1778.-   38. Zhou D, Ursitti J A, Bloch R J (1998). Developmental expression    of spectrins in rat skeletal muscle. Mol Biol Cell; 9:47-61.-   39. Li Z, Mericskay M, Agbulut O, Butler-Browne G, Carlsson L,    Thorne11 L E, et al (1997). Desmin is essential for the tensile    strength and integrity of myofibrils but not for myogenic-   40. Li Z L and Paulin D (1991). High level desmin expression depends    on a muscle-specific enhancer. J Biol Chem; 266:6562-6570.-   41. Hill C S, Duran S, Lin Z X, Weber K and Holtzer H (1986). Titin    and myosin, but not desmin, are linked during myofibrillogenesis in    postmitotic mononucleated myoblasts. J Cell Biol; 103:2185-2196.-   42. Sharp P S, Bye-a-Jee H, and Wells D J (2011). Physiological    characterization of muscle strength with variable levels of    dystrophin restoration in mdx mice following local antisense    therapy. Mol Ther; 19:165-171.-   43. Lazarides E (1980) Intermediate filaments as mechanical    integrators of cellular space. Nature; 283: 249-256.-   44. Lazarides W and Hubbard B D (1976) Immunological    characterization of the subunit of the 100 A filaments from muscle    cells. Proc Natl Acad Sci USA; 73: 4344-4348.-   45. Capetanaki Y, Milner D J, and Weitzer G (1997). Desmin in muscle    formation and maintenance: knockouts and consequences. Cell Struct    Funct.; 22:103-116.-   46. Bader D (1981). Density and distribution of    alpha-bungarotoxin-binding sites in postsynaptic structures of    regenerated rat skeletal muscle. J Cell Biol; 88:338-345.-   47. Ko P K, Anderson M J, and Cohen M W (1977). Denervated skeletal    muscle fibers develop discrete patches of high acetylcholine    receptor density. Science; 196:540-542.-   48. Nowack A, Yao J, Custer K L, and Bajjalieh S M (2010). SV2    regulates neurotransmitter release via multiple mechanisms. Am J    Physiol Cell Physiol; 299: C960-967.-   49. Vautrin J (2009). SV2 frustrating exocytosis at the    semi-diffusor synapse. Synapse; 63: 319-338.-   50. McLoughlin T J, Snyder A R, Brolinson P G, and Pizza F X (2004).    Sensory level electrical muscle stimulation: effect on markers of    muscle injury. Br J Sports Med; 38:725-729.-   51. Wang W J, Zhu H, Li F, Wan L D, Li H C, and Ding W L (2009).    Electrical stimulation promotes motor nerve regeneration selectivity    regardless of end-organ connection. J Neurotrauma; 26:641-649.-   52. Pedrotty D M, Koh J, Davis B H, Taylor D A, Wolf P, and Niklason    L E (2005). Engineering skeletal myoblasts: roles of    three-dimensional culture and electrical stimulation. Am J Physiol    Heart Circ Physiol; 288:H1620-1626.-   53. Stern-Straeter J, Bach A D, Stangenberg L, Foerster V T, Horch R    E, Stark G B, et al (2005). Impact of electrical stimulation on    three-dimensional myoblast cultures—a real-time R T-PCR study. J    Cell Mol Med; 9:883-892.-   54. Gerard C, Forest M A, Beauregard G, Skuk D, and Tremblay J P    (2012). Fibrin gel improves the survival of transplanted myoblasts.    Cell Transplant; 21:127-137.-   55. Cabianca, Cabianca D S, Casa V, Bodega B, Xynos A, Ginelli E,    Tanaka Y, Gabellini D. Cell. 2012 May 11; 149(4):819-31. doi:    10.1016/j.cell.2012.03.035. Epub 2012 Apr. 26.-   56. Cheli S, Francois S, Bodega B, Ferrari F, Tenedini E, Roncaglia    E, Ferrari S, Ginelli E, Meneveri R. PLoS One. 2011; 6(6):e20966.    doi: 10.1371/journal.pone.0020966. Epub 2011 Jun. 13.

1. A method of treating a subject having a muscle disorder comprisingthe steps of: a) identifying a subject having a muscle disorder in needof treatment; b) injecting human myogenic precursor cells in an amountcapable of forming mature muscle tissue into a portion of a limb of thesubject; c) subjecting a nerve of the limb to therapeutic stimulationconfigured to enhance engraftment of the human myogenic precursor cells;and d) creating a graft of the human myogenic precursor cells to promotegeneration of mature muscle tissue, wherein the generation of maturemuscle tissue improves muscle function.
 2. The method of claim 1,wherein the subject is a mammal.
 3. The method of claim 2, wherein ifthe subject is a non-human mammal, the non-human mammal isimmunocompromised and the limb is irradiated.
 4. The method of claim 2,wherein the mammal is a human.
 5. The method of claim 1, wherein thetherapeutic stimulation is therapeutic electrical stimulation.
 6. Themethod of claim 1, wherein engraftment is promoted by a means other thantherapeutic electrical stimulation.
 7. The method of claim 1, whereinengraftment is promoted by exercise.
 8. The method of claim 1, whereinthe muscle disease is selected from the group consisting of: acidmaltase deficiency (AMD), Andersen-Tawil Syndrome, Becker MuscularDystrophy (BMD), Becker Myotonia Congenita, Bethlem Myopathy,Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy), CarnitineDeficiency, Central Core Disease (CCD), Centronuclear Myopathy,Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD),Congenital Myotonic Dystrophy, Dejerine-Sottas Disease (DSD),Dermatomyositis (DM), Distal Muscular Dystrophy (DD), Duchenne MuscularDystrophy (DMD), Dystrophia Myotonica (Myotonic Muscular Dystrophy),Emery-Dreifuss Muscular Dystrophy (EDMD), Eulenberg Disease(Paramyotonia Congenita), Facioscapulohumeral Muscular Dystrophy (FSH orFSHD), Finnish (Tibial) Distal Myopathy, Friedreich's Ataxia (FA),Fukuyama Congenital Muscular Dystrophy, Glycogenosis Type 2,Glycogenosis Type 5, Glycogenosis Type 7, Glycogenosis Type 9,Gowers-Laing Distal Myopathy, Hauptmann-Thanheuser MD (Emery-DreifussMuscular Dystrophy), Hereditary Inclusion-Body Myositis, HereditaryMotor and Sensory Neuropathy (Charcot-Marie-Tooth Disease), HyperthyroidMyopathy, Hypothyroid Myopathy, Inclusion-Body Myositis (IBM), InheritedMyopathies, Integrin-Deficient Congenital Muscular Dystrophy, KennedyDisease (Spinal-Bulbar Muscular Atrophy), Kugelberg-Welander Disease(Spinal Muscular Atrophy), Lactate Dehydrogenase Deficiency,Lambert-Eaton Myasthenic Syndrome (LEMS), Limb-Girdle MuscularDystrophies (LGMDs), Lou Gehrig's Disease (Amyotrophic LateralSclerosis), Merosin-Deficient Congenital Muscular Dystrophy, MetabolicDiseases of Muscle, Mitochondrial Myopathy, Miyoshi Distal Myopathy,Motor Neurone Disease, Muscle-Eye-Brain Disease, Myasthenia Gravis (MG),Myofibrillar Myopathy, Myotonia Congenita (MC), Myotonic MuscularDystrophy (MMD), Myotubular Myopathy (MTM or MM), Nemaline Myopathy,Nonaka Distal Myopathy, Oculopharyngeal Muscular Dystrophy (OPMD),Paramyotonia Congenita, Periodic Paralysis, Peroneal Muscular Atrophy(Charcot-Marie-Tooth Disease), Pompe Disease (Acid Maltase Deficiency),Progressive External Ophthalmoplegia (PEO), Rod Body Disease (NemalineMyopathy), Spinal Muscular Atrophy (SMA), Spinal-Bulbar Muscular Atrophy(SBMA), Steinert Disease (Myotonic Muscular Dystrophy), Thomsen Disease(Myotonia Congenita), Ullrich Congenital Muscular Dystrophy,Walker-Warburg Syndrome (Congenital Muscular Dystrophy), Welander DistalMyopathy, and Werdnig-Hoffmann Disease (Spinal Muscular Atrophy).
 9. Themethod of claim 5, wherein the therapeutic electrical stimulation ispreferably intermittent neuromuscular electrical stimulation.